Dimeric compounds and their use as anti-viral agents

ABSTRACT

The invention relates to compounds of general formula (I); in which R is an amino or guanidino group; R 2  is acetyl or trifluoroacetyl; and n is an integer from 10 to 18 or a pharmaceutically acceptable derivative thereof, methods for their preparation, pharmaceutical formulations containing them or their use in the prevention or treatment of a viral infection

This invention relates to new chemical compounds and their use inmedicine. In particular the invention concerns novel dimeric compounds,methods for their preparation, pharmaceutical formulations thereof andtheir use as anti-viral agents.

BACKGROUND OF THE INVENTION

Enzymes with the ability to cleave N-acetyl neuraminic acid (NANA), alsoknown as sialic acid, from other carbohydrates are present in manymicroorganisms. These include bacteria such as Vibrio cholerae,Clostridium perfringens, Streptococcus pneumoniae and Arthrobactersialophilus, and viruses such as influenza virus, parainfluenza virus,mumps virus, Newcastle disease virus and Sendai virus. Most of theseviruses are of the orthomyxovirus or paramyxovirus groups, and carry aneuraminidase activity on the surface of the virus particles. Many ofthese neuraminidase-possessing organisms are major pathogens of manand/or animals, and some, such as influenza virus and Newcastle diseasevirus, cause diseases of enormous importance.

It has long been thought that inhibitors of neuraminidase might preventinfection by neuraminidase-bearing viruses. Most of the knownneuraminidase inhibitors are analogues of neuraminic acid, such as2-deoxy-2,3-dehydro-N-acetylneuraminic acid (DANA) and some of itsderivatives (Meindl et al, Virology, 1974 58 457). Our InternationalPatent Publication No. WO 91/16320 describes a number of analogues ofDANA which are active against viral neuraminidase, and it has been shownin particular that 4-guanidino-2-deoxy-2,3-dehydro-N-acetylneuraminicacid (Compound (A), code number GG167) is useful in the treatment ofinfluenza A and B (N. Engl. J. Med., 1997 337 874–880). Other patentapplications describe various closely-related sialic acid derivatives(eg. PCT Publications No. WO 95/18800, No. WO 95/20583 and No. WO98/06712), and anti-viral macromolecular conjugates of GG167 have alsobeen described (International Patent Application No. PCT/AU97/00771).

International Patent Publication No. WO 00/55149, describes dimericcompounds which comprise two neuraminidase binding molecules, such ascompound (A), attached to a common spacer or linking group of up to 100atoms in length.

We have now discovered a novel class of compounds which fall within thegeneric scope of International Patent Publication No. WO 00/55149, butwhich are not specifically disclosed therein, and exhibit a surprisinglyadvantageous anti-influenza activity profile which includes an enhancedlung residency time and high potency.

Without wishing to be bound by theory, the basis for the long residencytime in the lungs is thought to be due to the size and molecular weightof the compounds preventing entry through tight junctions in therespiratory epithelium and the polarity of the compounds being such thatpassage through the cell membranes occurs very inefficiently. Analternative theory is that the compounds themselves interact with thephospholipids in the cell membrane or other components of therespiratory epithelium and increase the residency time in the lungs.

SUMMARY OF THE INVENTION

In a first aspect, the present invention provides for a compound ofgeneral formula (I):

in which

R is an amino or guanidino group;

R² is acetyl or trifluoroacetyl; and

n is an integer from 10 to 18

or a pharmaceutically acceptable derivative thereof.

Preferably R is a guanidino group.

Preferably R² is an acetyl group.

Preferably n is 10 to 14, most preferably 12 to 14.

It will be appreciated by those skilled in the art that the compounds offormula (I) may be modified to provide pharmaceutically acceptablederivatives thereof at any one or more of the functional groups in thecompounds of formula (I). Of particular interest as such derivatives arecompounds modified at the carboxyl function, hydroxyl functions or atamino groups. Thus compounds of interest include alkyl esters, such asmethyl, ethyl, propyl or isopropyl esters, aryl esters, such as phenyl,benzoyl esters, and acetyl esters of the compounds of formula (I).

The term “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ether, ester or salt of such ester ofa compound of formula (I) or any other compound which, uponadministration to the recipient, is capable of providing a compound offormula (I) or an anti-virally active metabolite or residue thereof. Ofparticular interest as derivatives are compounds modified at the sialicacid carboxy or glycerol hydroxy groups, or at amino and guanidinegroups.

Pharmaceutically acceptable salts of the compounds of formula (I)include those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acids includehydrochloric, hydrobromic, sulphuric, nitric, perchloric, fumaric,maleic, phosphoric, glycollic, lactic, salicylic, succinic,toluene-p-sulphonic, tartaric, acetic, citric, methanesulphonic, formic,benzoic, malonic, naphthalene-2-sulphonic and benzenesulphonic acids.Other acids such as oxalic acid, while not in themselvespharmaceutically acceptable, may be useful in the preparation of saltsuseful as intermediates in obtaining compounds of the invention andtheir pharmaceutically acceptable acid addition salts.

Salts derived from appropriate bases include alkali metal (eg. sodium),alkaline earth metal (eg. magnesium), ammonium, and NR₄ ⁺ (where R isC₁₋₄alkyl) salts.

The compounds of the invention may be prepared by methods describedherein. It will be apparent to those skilled in the art, that it isnecessary to use protecting groups to protect one or more functionalgroups of the neuraminidase binding molecule during the process ofattaching the monomers to the alkyl spacer group. See for example“Protective Groups in Organic Synthesis” by T. W. Green and P. G. M.Nuts (John Wiley & Sons, 1991). Pharmaceutically acceptable salts of thecompounds of formula (I) may be prepared according to known procedures.

Accordingly, the present invention also provides a method for thepreparation of the compound of formula (I), which comprises the step ofdeprotection of a compound of formula (II)

in which n is as defined above, P₁ is a carboxylic acid protecting groupand P₂ is an amine protecting group.

The present invention further provides a method for the preparation ofthe compound of formula (I), which comprises the steps of:

(a) reacting a compound of formula (III)

in which P₁ and P₂ are as defined above, with a compound of formula(IV):OCN(CH₂)_(n)NCO  (IV)

in which n is as defined above to form the compound of formula (II) asdefined above; and

(b) deprotection of the compound of formula (II).

The present invention still further provides a method for thepreparation of the compound of formula (I) which comprises the steps of:

(a) protecting a compound of formula (V)

in which P₁ and P₂ are as defined above to form the compound of formula(III) as defined above;

(b) reacting the compound of formula (III) with the compound of formula(IV) as defined above to form the compound of formula (II) as definedabove; and

(c) deprotection of the compound of formula (II).

For use in therapy it is preferable that the compounds of formula (I)are in crystalline form. We have found that the compound of formula (I)in which R is a guanidino group, R² is an acetyl group and n is 13(Example 4 below) can be prepared in crystalline form by crystallisationfrom aqueous solution by procedures described herein.

The compounds of formula (I) possess antiviral activity. In particularthese compounds are inhibitors of viral neuraminidase oforthomyxoviruses and paramyxoviruses, for example the viralneuraminidase of influenza A and B, parainfluenza, mumps and Newcastledisease.

Thus in a second aspect the invention provides a compound of formula (I)or a pharmaceutically acceptable derivative thereof, for use as anactive therapeutic agent in the treatment of a viral infection, forexample orthomyxovirus and paramyxovirus infections.

In a third aspect the invention provides a method for the prevention ortreatment of a viral infection comprising the step of administration toa subject in need thereof of an effective amount of a compound offormula (I), or a pharmaceutically acceptable salt or derivativethereof.

Preferably, the viral infection is an orthomyxovirus or paramyxovirusinfection. More preferably the viral infection is an influenza A or Binfection.

Preferably the subject is an animal such as a mammal, more preferably ahuman, or a member of the genus Equus, for example a horse, donkey ormule. Most preferably the mammal is a human.

In a fourth aspect the invention provides use of a compound of theinvention for the manufacture of a medicament for the treatment of aviral infection.

As used herein, the term “effective amount” is meant an amount of thecompound of formula I effective to preventing or treating a viralinfection in order to yield a desired therapeutic response. For example,to overcome or alleviate the effects of a viral infection.

The term “therapeutically-effective amount” means an amount of thecompound of formula I to yield a desired therapeutic response. Forexample, treating or preventing a viral infection.

The specific “therapeutically-effective amount” will, obviously, varywith such factors as the particular viral infection being treated, thephysical condition of the subject, the type of animal being treated, theduration of the treatment, the nature of concurrent therapy (if any),and the specific formulation employed and the structure of the compoundor its derivatives.

Generally, the terms “treating”, “treatment” and the like are usedherein to mean affecting a subject, tissue or cell to obtain a desiredpharmacologic and/or physiologic effect. The effect may be prophylacticin terms of completely or partially preventing a viral infection or signor symptom thereof, and/or may be therapeutic in terms of a partial orcomplete cure of a viral infection. “Treating” as used herein covers anytreatment of, or prevention of a viral infection in a vertebrate, amammal, particularly a human, and includes: (a) preventing the viralinfection from occurring in a subject that may be predisposed to theviral infection, but has not yet been diagnosed to the viral infection,but has not yet been diagnosed as having it; (b) inhibiting the viralinfection, ie., arresting its development; or (c) relieving orameliorating the effects, i.e., cause regression of the symptoms of theviral infection.

The compounds of the invention may also be used in diagnostic methods,in particular methods for the detection of influenza virus. For use insuch methods it may be advantageous to link a compound of the inventionto a label, such as a radioactive, fluorescent or chemiluminescentlabel.

Methods of diagnosis for which the compounds of the invention aresuitable are described, for example, in our earlier applicationsPCT/AU97/00109 and PCT/AU97/00771.

In a fifth aspect the invention provides a method for the detection of aviral infection which comprises the step of contacting the compound ofthe invention with a sample suspected of containing the virus.

It will be further appreciated that the amount of a compound of theinvention required for use in treatment will vary not only with theparticular compound selected but also with the route of administration,the nature of the condition being treated, and the age and condition ofthe patient, and will ultimately be at the discretion of the attendantphysician or veterinarian. In general however, a suitable dose will bein the range of from about 0.001 to 100 mg/kg of bodyweight per day,preferably in the range of 0.01 to 10 mg/kg/day, most preferably in therange of 0.1 to 1 mg/kg/day.

Treatment is preferably commenced before or at the time of infection andcontinued until virus is no longer present in the respiratory tract.However the compounds are also effective when given post-infection, forexample after the appearance of established symptoms.

Suitably treatment is given on one or two occasions, preferably onlyonce only for treatment, and preferably once per week for prophylaxis.

The compound is conveniently administered in unit dosage form, forexample containing 1 to 100 mg, more conveniently 1 to 20 mg of activeingredient per unit dosage form.

While it is possible that, for use in therapy, a compound of theinvention may be administered as the raw chemical, it is preferable topresent the active ingredient as a pharmaceutical formulation.

Thus in a sixth aspect the invention provides a pharmaceuticalformulation comprising a compound of formula (I) or a pharmaceuticallyacceptable salt or derivative thereof, together with one or morepharmaceutically acceptable carriers therefor and, optionally, othertherapeutic and/or prophylactic ingredients. The carrier(s) must be“acceptable” in the sense of being compatible with the other ingredientsof the formulation and not being deleterious to the recipient thereof.

The compounds of the invention may also be used in combination withother therapeutic and/or prophylactic agents, for example otheranti-infective agents. In particular the compounds of the invention maybe employed with other antiviral agents. The invention thus provides ina seventh aspect a combination comprising a compound of formula (I) or apharmaceutically acceptable salt or derivative thereof together withanother therapeutically and/or prophylactically active agent, inparticular an antiviral agent.

The combinations referred to above may conveniently be presented for usein the form of a pharmaceutical formulation and thus such formulationscomprising a combination as defined above together with apharmaceutically acceptable carrier therefor comprise a further aspectof the invention.

Suitable therapeutic and/or prophylactic agents for use in suchcombinations include other anti-infective agents, in particularanti-bacterial and anti-viral agents such as those used to treatrespiratory infections. For example, other compounds or vaccineseffective against influenza viruses, such as the sialic acid analoguesreferred to above, e.g. zanamivir, oseltamivir, amantadine, rimantadineand ribavirin and FluVax, may be included in such combinations.

The individual components of such combinations may be administeredeither separately, sequentially or simultaneously in separate orcombined pharmaceutical formulations.

When the compounds of the invention are used with a second therapeuticand/or prophylactic agent active against the same virus, the dose ofeach compound may either be the same as or different from that employedwhen each compound is used alone. Appropriate doses will be readilyappreciated by those skilled in the art.

Pharmaceutical formulations include those suitable for oral, rectal,nasal, topical (including buccal and sub-lingual), vaginal or parenteral(including intramuscular, sub-cutaneous and intravenous) administration,or those in a form suitable for administration to the respiratory tract(including the nasal passages) for example by inhalation orinsufflation. The formulations may, where appropriate, be convenientlypresented in discrete dosage units, and may be prepared by any of themethods well known in the art of pharmacy. These methods include thestep of bringing into association the active compound with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Pharmaceutical formulations suitable for oral administration mayconveniently be presented as discrete units such as capsules, cachets ortablets each containing a predetermined amount of the active ingredient;as a powder or granules; as a solution, a suspension or as an emulsion.The active ingredient may also be presented as a bolus, electuary orpaste. Tablets and capsules for oral administration may containconventional excipients such as binding agents, fillers, lubricants,disintegrants, or wetting agents. The tablets may be coated according tomethods well known in the art. Oral liquid preparations may for examplebe in the form of aqueous or oily suspensions, solutions, emulsions,syrups or elixirs, or may be presented as a dry product for constitutionwith water or other suitable vehicle before use. Such liquidpreparations may contain conventional additives such as suspendingagents, emulsifying agents, non-aqueous vehicles, which may includeedible oils, or preservatives.

The compounds according to the invention may also be formulated forparenteral administration by injection, for example bolus injection, orcontinuous infusion, and may be presented in unit dose form in ampoules,pre-filled syringes, small volume infusion or in multi-dose containerswith an added preservative. The compositions may take such forms assuspensions, solutions, or emulsions in oily or aqueous vehicles, andmay contain formulating agents such as suspending, stabilising and/ordispersing agents. Alternatively, the active ingredient may be in powderform, obtained by aseptic isolation of sterile solid or bylyophilisation from solution, for constitution with a suitable vehicle,eg. sterile, pyrogen-free water, before use.

For topical administration to the epidermis the compounds according tothe invention may be formulated as ointments, creams or lotions, or as atransdermal patch. Ointments and creams may, for example, be formulatedwith an aqueous or oily base with the addition of suitable thickeningand/or gelling agents. Lotions may be formulated with an aqueous or oilybase, and will in general also contain one or more emulsifying agents,stabilising agents, dispersing agents, suspending agents, thickeningagents, or colouring agents.

Formulations suitable for topical administration in the mouth includelozenges comprising active ingredient in a flavoured base, usuallysucrose and gum acacia or gum tragacanth; pastilles comprising theactive ingredient in an inert base such as gelatin or sucrose and gumacacia; and mouthwashes comprising the active ingredient in a suitableliquid carrier.

Pharmaceutical formulations suitable for rectal administration whereinthe carrier is a solid are most preferably presented as unit dosesuppositories. Suitable carriers include cocoa butter and othermaterials commonly used in the art, and the suppositories may beconveniently formed by admixture of the active compound with thesoftened or melted carrier(s) followed by chilling and shaping moulds.

Formulations suitable for vaginal administration may be presented aspessaries, tampons, creams, gels, pastes, foams or sprays containing inaddition to the active ingredient such carriers as are known in the artto be appropriate.

For administration to the respiratory tract, including intranasaladministration, the neuraminidase inhibitors may be administered by anyof the methods and formulations employed in the art for administrationto the respiratory tract.

Thus in general the compounds may be administered in the form of asolution or a suspension or as a dry powder.

Solutions and suspensions will generally be aqueous, for exampleprepared from water alone (for example sterile or pyrogen-free water) orwater and a physiologically acceptable co-solvent (for example ethanol,propylene glycol or polyethylene glycols such as PEG 400).

Such solutions or suspensions may additionally contain other excipientsfor example preservatives (such as benzalkonium chloride), solubilisingagents/surfactants such as polysorbates (eg. Tween 80, Span 80,benzalkonium chloride), buffering agents, isotonicity-adjusting agents(for example sodium chloride), absorption enhancers and viscosityenhancers. Suspensions may additionally contain suspending agents (forexample microcrystalline cellulose, carboxymethyl cellulose sodium).

Solutions or suspensions are applied directly to the nasal cavity byconventional means, for example with a dropper, pipette or spray. Theformulations may be provided in single or multidose form. In the lattercase a means of dose metering is desirably provided. In the case of adropper or pipette this may be achieved by the patient administering anappropriate, predetermined volume of the solution or suspension. In thecase of a spray this may be achieved for example by means of a meteringatomising spray pump.

Administration to the respiratory tract may also be achieved by means ofan aerosol formulation in which the compound is provided in apressurised pack with a suitable propellant, such as achlorofluorocarbon (CFC), for example dichlorodifluoromethane,trichlorofluoromethane or dichlorotetrafluoroethane, carbon dioxide orother suitable gas. The aerosol may conveniently also contain asurfactant such as lecithin. The dose of drug may be controlled byprovision of a metered valve.

Alternatively the compounds may be provided in the form of a dry powder,for example a powder mix of the compound in a suitable powder base suchas lactose, starch, starch derivatives such as hydroxypropylmethylcellulose and polyvinylpyrrolidine (PVP). Conveniently the powdercarrier will form a gel in the nasal cavity. The powder composition maybe presented in unit dose form, for example in capsules or cartridges ofeg. gelatin, or blister packs from which the powder may be administeredby means of an inhaler.

In formulations intended for administration to the respiratory tract,including intranasal formulations, the compound will generally have asmall particle size, for example of the order of 5 microns or less. Sucha particle size may be obtained by means known in the art, for exampleby micronisation.

When desired, formulations adapted to give sustained release of theactive ingredient may be employed.

Preferably the compounds of the invention are administered to therespiratory tract by inhalation, insufflation or intranasaladministration, or a combination thereof.

“Relenza” is administered by oral inhalation as a free-flow powder via a“Diskhaler” (trade marks of the GlaxoSmithKline group of companies). Asimilar formulation would be suitable for the present invention.

Thus, according to an eighth aspect of the present invention there isprovided an inhaler which contains a formulation as defined above.

It will be appreciated that the inhaler may also be in the form of ameter dose aerosol inhaler.

For the purposes of this specification it will be clearly understoodthat the word “comprising” means “including but not limited to”, andthat the word “comprises” has a corresponding meaning.

All publications, including but not limited to patents and patentapplications, cited in this specification are herein incorporated byreference as if each individual publication were specifically andindividually indicated to be incorporated by reference herein as thoughfully set forth.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference onlyto the following non-limiting examples.

Machine Methods

Method A (LC/MS)

-   Micromass Platform II mass spectrometer operating in positive ion    electrospray mode, mass range 100–1000 amu.-   Column: 3.3 cm×4.6 mm ID, 3 μm ABZ+PLUS-   Flow Rate: 3 ml/min-   Injection Volume: 5 μl-   Solvent A: 95% acetonitrile+0.05% formic acid-   Solvent B: 0.1% formic acid+10 mMolar ammonium acetate-   Gradient: 0–100% A/5 min, 100–0% B/5 min    Method B-   The prep column used was a Supelcosil ABZplus(10 cm×2.12 cm).-   UV wavelength: 230 nm-   Flow: 4 ml/min-   Solvent A: acetonitrile+0.05% TFA-   Solvent B: water+0.1% TFA-   Gradient: 20–40% A/20 min, 40% A/20 min, 40–100% A/0.3 min, 100%    A/15 min, 100–20% A/3 min    Abbreviations-   TFA trifluoroacetic acid-   DMAP 4-dimethylaminopyridine-   SPE solid phase extraction-   DPM diphenylmethane-   BOC t-butoxycarbonyl group    Preparation of Intermediate 1

Benzhydryl(2R,3R,4S)-3-(acetylamino)-4-({(E)-[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-[(1R,2R)-1,2,3-trihydroxypropyl]-3,4-dihydro-2H-pyran-6-carboxylate(see J. Med. Chem. 1998, 41, 787–797) (12.38 g; 17.7 mmoles) wasdissolved in dry acetonitrile (130 ml) under nitrogen at roomtemperature. The solution was stirred and 1,1′-carbonyldiimidazole (2.87g; 17.7 mmoles) was added. After 16 hours LC/MS showed the presence ofstarting triol so further 1,1′-carbonyldiimidazole (total of 0.493 g; 3mmoles) was added. After a few hours LC/MS showed no triol present. Thesolvent was evaporated and the residue flash columned on silica, elutingwith 1:1 ethyl acetate/40–60 petroleum ether. Fractions containingwanted product were evaporated then taken up in dichloromethane, driedwith sodium sulphate, filtered and evaporated to give Intermediate 1(benzhydryl(2R,3R,4S)-3-(acetylamino)-4-({[(tert-butoxycarbonyl)amino][(tert-butoxycarbonyl)imino]methyl}amino)-2-{(S)-hydroxy[(4R)-2-oxo-1,3-dioxolan-4-yl]methyl}-3,4-dihydro-2H-pyran-6-carboxylate)as an off white solid (11.05 g; 86%).

Preparation of Intermediate 10

Intermediate 1 (0.4 g; 0.56 mmole) was dissolved in dry dichloromethane(0.5 ml). To this was added DMAP (20 mg) and 4 molecular sieves type 3Afollowed by intermediate 5 (50 mg; 0.19 mmole). The mixture was refluxedovernight then applied directly to a 10 g Si SPE cartridge eluted withdiethyl ether and ethyl acetate to give intermediate 10 as a colourlessglass (0.16 g, 50% yield).

LC/MS (method A) showed (M+2H⁺)/2=858; T_(RET)=4.68 min.

Similarly prepared were the following:

n diisocyanate dicarbamate (M + 2H⁺)/2 T_(RET)(min) 10 intermediate 2intermediate 7 837 4.58 11 intermediate 3 intermediate 8 844 4.68 12intermediate 4 intermediate 9 851 4.66 14 intermediate 6 intermediate 11865 4.75Preparation of Intermediate 15

Intermediate 10 (0.16 g; 0.093 mmole) was dissolved in a 10:1 mixture ofdichloromethane: anisole (6.3 ml) at room temperature. To this was addedTFA (6.3 ml) and the resulting solution was stirred for 2.5 hours thenevaporated in vacuo. Trituration of the residue with ether gaveintermediate 15 as the di-TFA salt (92 mg; 82% yield). LC/MS (method A)showed (M+2H⁺)/2=492; T_(RET)=2.61 min.

Similarly prepared were the following:

starting n material product (M + 2H⁺)/2 T_(RET)(min) 10 intermediate 7intermediate 12 471 2.31 11 intermediate 8 intermediate 13 478 2.43 12intermediate 9 intermediate 14 485 2.51 14 intermediate 11 intermediate16 499 2.68

EXAMPLE 4 n=13(2R,3R,4S)-3-(acetylamino)-2-{(1R,21R,22R)-21-((2R,3R,4S)-3-(acetylamino)-4-{[amino(imino)methyl]amino}-6-carboxy-3,4-dihydro-2H-pyran-2-yl)-1-[(1R)-1,2-dihydroxyethyl]-22,23-dihydroxy-3,19-dioxo-2,20-dioxa-4,18-diazatricos-1-yl}-4-{[amino(imino)methyl]amino}-3,4-dihydro-2H-pyran-6-carboxylicacid bis(trifluoroacetic acid salt)

Intermediate 15 (92 mg; 0.076 mmole) was dissolved in a mixture of water(16 ml) and methanol (16 ml). To this was added triethylamine (4 ml) andthe solution was stirred for 1 hour. Volatile organics were removed invacuo and the residue adjusted to pH 2 with TFA. Reverse phasepreparative HPLC (method B) gave example 4 as the di-TFA salt (35.5 mg;40% yield). LC/MS (method A) showed (M+2H⁺)/2=466; T_(RET)=2.45 min.

Elemental analysis:—Found: C, 42.00; H, 5.79; N, 11.00%. Calc fortetrahydrate: C, 41.95; H, 6.18; N, 11.38%. NMR(D₂O) δ: 5.85 (2H, d,2×CH); 4.85 (2H, dd, 2×CH); 4.46 (2H, dd, 2×CH); 4.34 (2H, dd, 2×CH);4.05, 2H, t, 2×CH); 3.94 (2H, m, 2×CH); 3.58 (2H, d d, CH₂); 3.42 (2H,dd, CH₂); 2.95 (4H, m, 2×CH₂); 1.88 (6H, s, 2×CH₃); 1.38 (4H, br.m,2×CH₂); 1.22–1.10 (18H, br.m, 9×CH₂) p.p.m.

EXAMPLE 4a Large Scale Preparation of Example 4

Intermediate 15 (2.8 g; 2.3 mmoles) was dissolved in water (50.4 ml). Tothis was added methanol (50.4 ml) followed by triethylamine (6.4 ml; 46mmoles). The resulting solution was stirred at room temperature for 5hours, the volume of the reaction mixture was reduced by ca 33% in vacuoat 35 degrees C. then the pH was adjusted to 2 with TFA (0.5 ml). Theacidified solution was then injected onto a Prochom LC50 HPLC systemcomprising of a 20 cm×5 cm column packed with 7 micron Kromasil C8packing material. The column was subjected to gradient elution:

-   Solvent A: water+1% TFA-   Solvent B: 75% acetonitrile/water+1% TFA-   Flow: 80 ml/min-   Gradient: 0% B to 100% B/40 min

The appropriate fractions were combined and the acetonitrile was removedin vacuo at 35 degrees C. The aqueous residue was absorbed onto a 10cm×22 mm column of Amberchrom CG-161 (PSDVB resin) and the column waswashed with water then eluted with acetonitrile:MeOH:water 2:2:1 (500ml). The solvent was removed in vacuo to yield a gum. The addition ofisopropanol (20 ml) gave a solid which was dried to give the product asthe zwitterion (1.68 g).

EXAMPLE 4b Crystallisation of Example 4

The zwitterion (100 mg; 0.1075 mmoles) was dissolved in water (35 ml).To this was added sodium bicarbonate (18.06 mg; 0.215 mmoles) and theresulting solution was freeze-dried to give a white solid. A sample (2mg) of this solid was dissolved in water (0.8 ml) and evaporated to asyrupy oil. Dioxan (1 ml) was added and a white solid formed. The solidwas allowed to settle and the supernatent was removed. Further dioxan (1ml) was added and the supernatant was again removed. This process wasrepeated twice more and the solid obtained was dried in vacuo.Examination under polarised light showed crystallinity.

Examples E1, E2, E3 and E5 were prepared using an analogous procedure tothat of Example E4.

EXAMPLE 1 n=10(2R,3R,4S)-3-(acetylamino)-2-{(1R,18R,19R)-18-((2R,3R,4S)-3-(acetylamino)-4-{[amino(imino)methyl]amino}-6-carboxy-3,4-dihydro-2H-pyran-2-yl)-1-[(1R)-1,2-dihydroxyethyl]-19,20-dihydroxy-3,16-dioxo-2,17-dioxa-4,15-diazaicos-1-yl}-4-{[amino(imino)methyl]amino}-3,4-dihydro-2H-pyran-6-carboxylicacid bis(trifluoroacetic acid salt)

LC/MS (method A) showed (M+2H⁺)/2=445; T_(RET)=2.13 min.

EXAMPLE 2 n=11(2R,3R,4S)-3-(acetylamino)-2-{(1R,19R,20R)-19-((2R,3R,4S)-3-(acetylamino)-4-{[amino(imino)methyl]amino}-6-carboxy-3,4-dihydro-2H-pyran-2-yl)-1-[(1R)-1,2-dihydroxyethyl]-20,21-dihydroxy-3,17-dioxo-2,18-dioxa-4,16-diazahenicos-1-yl}-4-{[amino(imino)methyl]amino}-3,4-dihydro-2H-pyran-6-carboxylicacid bis(trifluoroacetic acid salt)

LC/MS (method A) showed (M+2H⁺)/2=452; T_(RET)=2.25 min.

EXAMPLE 3 n=12(2R,3R,4S)-3-(acetylamino)-2-{(1R,20R,21R)-20-((2R,3R,4S)-3-(acetylamino)-4-{[amino(imino)methyl]amino}-6-carboxy-3,4-dihydro-2H-pyran-2-yl)-1-[(1R)-1,2-dihydroxyethyl]-21,22-dihydroxy-3,18-dioxo-2,19-dioxa-4,17-diazadocos-1-yl}-4-{[amino(imino)methyl]amino}-3,4-dihydro-2H-pyran-6-carboxylicacid bis(trifluoroacetic acid salt)

LC/MS (method A) showed (M+2H⁺)/2=459; T_(RET)=2.34 min.

EXAMPLE 5 n=14(2R,3R,4S)-3-(acetylamino)-2-{(1R,22R,23R)-22-((2R,3R,4S)-3-(acetylamino)-4-{[amino(imino)methyl]amino}-6-carboxy-3,4-dihydro-2H-pyran-2-yl)-1-[(1R)-1,2-dihydroxyethyl]-23,24-dihydroxy-3,20-dioxo-2,21-dioxa-4,19-diazatetracos-1-yl)}-4-{[amino(imino)methyl]amino}-3,4-dihydro-2H-pyran-6-carboxylicacid bis(trifluoroacetic acid salt)

LC/MS (method A) showed (M+2H⁺)/2=473; T_(RET)=2.50 min.

EXAMPLE 6 Evaluation of the Compounds of Formula (I)—Inhibition ofInfluenza Virus Replication

Cytopathic effect (CPE) assays were performed essentially as describedby Watanabe et al. (J. Virological Methods, 1994 48 257). MDCK cellswere infected with a defined inoculum of virus (determined byexperimentation to be the minimum sufficient to cause adequate CPE in 72hours and to be susceptible to control compounds at concentrationsconsidered to be consistent with published norms) in the presence serialdilutions of Compounds of the invention. Cultures were incubated for upto 72 hours at 37° C. in a 5% CO₂ atmosphere. The extent of CPE andhence viral replication was determined via metabolism of the viral dye3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)according to published methods (see for example, Watanabe et al., 1994).The compound concentration that inhibited CPE by 50% (ID₅₀) wascalculated using a computer program for curve fitting. InfluenzaA/Sydney/5/97 and B/Harbin/7/95 viruses were assayed and the results areshown in Table 1. Comparable data for a specifically disclosed compoundin WO 00/55149 and for compound A is also shown in Table 1.

TABLE 1 ID₅₀ μg/ml ID₅₀ M ID₅₀ μg/ml ID₅₀ M Description A/Sydney/5/97+A/Sydney/5/97+ B/Harbin/7/95 B/Harbin/7/95 Compound A 0.023 +/− 0.024 690.013 +/− 0.011 39 E1 0.0002 0.179 0.0001 0.09 E2 0.0001 0.09 0.00010.09 E3 0.0001, 0.0001 0.087 0.0001, 0.0001 0.087 E4 0.0001 0.086 0.00010.086 E5 0.0001 0.085 0.0003 0.26 Compound 0.0007, 0.0005 0.58, 0.750.007 +/− 0.01 5.8 Number 8* Compound 0.057 66 >0.1 >115 Number 10* *Asreferenced in WO 00/55149 +Data provided in WO 00/55149 related to thevirus H3N2 isolate A/Victoria/3/75 rather than A H3N2 isolateA/Sydney/5/97. When comparing such data the person skilled in the artwill appreciate that differences in antiviral potency are not uncommonfor a given compound when analysed against several different viruses invitro. For example, Woods et al (Antimicrob Agents Chemother 1993 37:1473–9) have reported that Compound A exhibits a wide range ofEC50values (from 0.02 to 0.16 uM) in in vitro assays involving recentclinical isolates. Accordingly, compound 8 was found to be more potentin CPE assays involving the recent influenza A H3N2 isolateA/Sydney/5/97 than the earlier H3N2 isolate A/Victoria/3/75.

Data provided in Table 1 demonstrate that the compounds E1–E5, inaddition to being substantially more potent than the highly activecompound A, are even more potent against A/Sydney/5/97 and substantiallymore potent against the recent influenza B isolate B/Harbin/7/95 thancompounds 8 and 10 of WO 00/55149.

EXAMPLE 7 Plaque Reduction Assay

Madin Darby Canine Kidney (MDCK) cells are seeded into six well tissueculture plates and grown to confluency via standard methods. Influenzaviruses are diluted in a minimal volume of phosphate buffered salinesupplemented with 0.2% bovine serum albumin to yield an estimated titreof 50–100 plaque forming units (pfu) per well. After adsorption to theMDCK cells for one hour at 37° C. in a 5% CO₂ atmosphere the viralinocula is aspirated and replaced with viral growth media (minimalEagle's media supplemented with BSA, trypsin andinsulin/transferrin/selenium at optimal concentrations) containingsufficient agar or agarose (generally 1–2%) to cause the media to gel atroom temperature and at 37° C. in a 5% CO₂ atmosphere until plaquesdevelop (generally 2–4 days). Plaques can be visualised with a suitablestain (e.g. 0.4% crystal violet in formal saline) before counting.Antiviral potency is expressed as the concentration of test articlewhich reduces plaque numbers by 50% of the untreated control value(EC₅₀).

EC₅₀ ng/ml PRA Example A/WSN* A/Vic* A/Syd* A/New* A/Pan* A/Bay*Compound A 56, >100 5.5 +/− 8.2 2.4 0.27, 0.23 2.7, 3 35 3 0.00230.000429 2 0.06, 0.2 <0.0001 4 <0.0001 <0.001, <0.01, 0.2 <0.0001 0.043<0.00001 5 <0.0001 <0.001, 0.02, 0.3 0.032 <0.0001 0.032 <0.0001Amantadine 220 11 157 Oseltamivir 0.11 0.23 0.3 *A/WSN/33 BVLV09 (H1N1)A/Victoria/3/75 BVLV017 (H3N2) A/Sydney/5/97 BVLV015 (H3N2) A/NewCaledonia/20/99 BVLV008 (H1N1) A/Panama/2007/99 BVLV008 (H3N2)A/Bayern/7/95 BVL006 (H1N1)

EC₅₀ ng/ml PRA B/ Example B/Vic* Harb* B/HongK* B/Yam* Compound A  3, 200.19 21 +/− 6 0.2, 3.1 3 0.009, 0.01  <0.0001 <0.0001, <0.0001 2 0.04,0.05 <0.0001 4 0.01, 0.1  0.06 <0.0001 5 0.05, 0.1  0.37 <0.0001Amantadine >10000 2061 Oseltamivir 32 0.7 *B/Victoria/1/67 B/HongKong/5/72 BVLV012 B/Harbin/7/95 BVLV008 B/Yamanashi/166/98 BVLV007

EXAMPLE 8 Assessment of Long Duration of Action

Rodents are anaesthetised and dosed with compound of interest by theintra-tracheal route at a dose volume of 0.8 ml/kg. The rodent is thenheld in the vertical position until full recovery is achieved. Atdifferent time points, for example, 2, 8, 24 and 48 hours post-dose,level of compound in the lung tissue are assessed by analytical methods.Any analytical method suitable for detection of this type of compoundmay be used. The time at which levels of compound fall below thesensitivity of the analytical techniques identified will determine theresidency time of the compound in lung tissue.

The rat lung retention data for selected compounds is shown below.Please note that all experiments included a co-dosed internal standard,namely compound 3 of International Patent Publication No. WO 02/20514,to permit comparison. The data are expressed as a ratio with respect tothis compound, the structure of which is shown below.

The data for compound A is included for comparison purposes. Thecompounds of the invention have significantly greater retention at 7days than Compound A when expressed as a ratio of compound concentrationto standard concentration.

Rat lung retention assay results Ratio Mean Mean Mean (lung) (cmpd)/PCTtime point dose (cmpd) (cmpd) PCT AU01/01128 (PCT AU01/01128 AU01/01128hrs Compound mg/kg ng/g ng/g compound 3 ng/g compound 3) ng/g compound 348 Example 3 0.1 591 1117 655 1413 0.79 48 Example 3 0.1 1845 1840 48Example 3 0.1 914 1744 168 Example 3 0.1 111 376 242 550 0.68 168Example 3 0.1 471 580 168 Example 3 0.1 546 829 48 Example 4 0.1 24141772 1098 1044 1.70 48 Example 4 0.1 1927 1352 48 Example 4 0.1 977 681168 Example 4 0.1 929 756 636 509 1.48 168 Example 4 0.1 914 524 168Example 4 0.1 426 367 48 Example 5 0.1 3044 4803 784 1478 3.25 48Example 5 0.1 6268 2046 48 Example 5 0.1 5097 1605 168 Example 5 0.12750 1798 632 363 4.95 168 Example 5 0.1 1255 242 168 Example 5 0.1 1388216 48 Compound A (zanamivir) 0.1 421 352 698 1368 0.26 48 Compound A(zanamivir) 0.1 369 1901 48 Compound A (zanamivir) 0.1 267 1507 168Compound A (zanamivir) 0.1 91 61 815 750 0.08 168 Compound A (zanamivir)0.1 47 925 168 Compound A (zanamivir) 0.1 45 512

EXAMPLE 9 Alternative Assessment of Long Duration of Action and Efficacy

The protocol for infecting mice has been described previously (1–4).Mildly anaesthetised mice are inoculated into the external nares withinfluenza virus. Treatment procedure and regimen. A single dose ofcompound is administered at a defined time point up to 10 days prior toinfection, preferably 4–7 days prior to infection, or followinginfection, preferably immediately following infection and up to 48 hourspost infection. In most experiments, a non-lethal strain of influenza isused, and efficacy is assessed by reductions in lung virus titre. Formice given compound prior to infection, lungs are removed post infectioneither on a single day, or on days following infection, preferably days1–4 post infection. Homogenised lung samples are assayed for virus usingestablished methods, and the titres of viral load estimated and comparedto titres of virus in lungs of untreated mice.

In those experiments where a mouse-adapted lethal strain of influenza isused, efficacy is assessed by an increase in survival rate and/ornumbers of survivors, as compared to untreated mice.

REFERENCES

-   1. Ryan, D. M., J. Ticehurst, M. H. Dempsey, and C. R. Penn, 1994.    Inhibition of influenza virus replication in mice by GG167    (4-guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid) is    consistent with extracellular activity of viral neuraminidase    (sialidase). Antimicrob. Agents and Chemother. 38 (10):2270–2275.-   2. von Itzstein M., W. -Y. Wu, G. B. Kok, M. S. Pegg, J. C.    Dyason, B. Jin, T. V. Phan, M. L. Smythe, H. F. White, S. W.    Oliver, P. M. Colman, J. N. Varghese, D. M. Ryan, J. M. Woods, R. C.    Bethell, V. J. Hogham, J. M. Cameron, and C. R. Penn. 1993. Rational    design of potent sialidase-based inhibitors of influenza virus    replication. Nature (London) 363:418–423.-   3. Woods, J. M., R. C. Bethell, J. A. V. Coates, N. Healey, S. A.    Hiscox, B. A. Pearson, D. M. Ryan, J. Ticehurst, J. Tilling, S. A.    Walcott, and C. R. Penn. 1993.    4-Guanidino-2,4-dideoxy-2,3-dehydro-N-acetylneuraminic acid is a    highly effective inhibitor both of the sialidase (neuraminidase) and    of growth of a wide range of influenza A and B viruses in vitro.    Antimicrob. Agents Chemother. 37:1473–1479.-   4. Robert J Fenton, Peter J Morley, Ian J Owens, David Gower, Simon    Parry, Lee Crossman and Tony Wong (1999). Chemoprophylaxis of    influenza A virus infections, with single doses of zanamivir,    demonstrates that zanamivir is cleared slowly from the respiratory    tract. Antimicrob. Agents and Chemother. 43, 11, 2642–2647.

1. A compound of general formula (I):

in which R is an amino or guanidino group; R² is acetyl ortrifluoroacetyl; and n is an integer from 10 to 14, or apharmaceutically acceptable salt, ether, ester or salt of such esterthereof.
 2. A compound according to claim 1, in which R is a guanidinogroup.
 3. A compound according to claim 1, in which R² is an acetylgroup.
 4. A compound according to claim 1, in which n is 12 to
 14. 5. Acompound according to claim 1, which contains a pharmaceuticallyacceptable salt, ether, ester or salt of such ester at one or more ofthe carboxyl groups, hydroxyl groups, amino groups or guanidine groups.6. A compound according to claim 1, in which said compound is an alkylester, an aryl ester or an acetyl ester.
 7. A method for the preparationof the compound of formula (I) according to claim 1, which comprises thestep of deprotecting a compound of formula (II)

in which n is as defined in claim 1, P₁ is a carboxylic acid protectinggroup and P₂ is an amine protecting group.
 8. A method for thepreparation of the compound of formula (I) according to claim 1, whichcomprises the steps of; (a) reacting a compound of formula (III)

 in which P₁ is a carboxylic acid protecting group and P₂ is an amineprotecting group, with a compound of formula (IV):OCN(CH₂)_(n)NCO  (IV)  in which n is as defined in claim 1, to form thecompound of formula (II)

 in which n is as defined in claim 1, P₁ is a carboxylic acid protectinggroup and P₂ is an amine protecting group; and (b) deprotecting thecompound of formula (II).
 9. A method for the preparation of thecompound of formula (I) according to claim 1, which comprises the stepsof: (a) protectinig a compound of formula (V)

 in which P₁ is a carboxylic acid protecting group and P₂ is an amineprotecting group to form the compound of formula (III)

 in which P₁ is a carboxylic acid protecting group and P₂ is an amineprotecting group; (b) reacting the compound of formula (III) with thecompound of formula (IV)OCN(CH₂)_(n)NCO  (IV)  in which n is as defined in claim 1 to form thecompound of formula (II)

 in which n is as defined in claim 1, P₁ is a carboxylic acid protectinggroup and P₂ is an amine protecting group.; and (c) deprotecting thecompound of formula (II).
 10. A pharmaceutical formulation comprising atherapeutically effective amount of a compound of formula (I) as definedin claim 1 or a pharmaceutically acceptable salt, ether, ester or saltof such ester thereof, together with one or more pharmaceuticallyacceptable carriers.
 11. A pharmaceutical formulation according to claim10, which further comprises one or more anti-viral agents used to treatrespiratory infections.
 12. A pharmaceutical formulation according toclaim 11, in which the agent is zanamivir, oseltamivir, amantadine,rimantadine, and/or ribavirin.
 13. An inhaler which comprises a compoundaccording to claim
 1. 14. An inhaler according to claim 13 which isadapted for oral administration as a free-flow powder.
 15. An inhaleraccording to claim 13 which is a metered dose aerosol inhaler.
 16. Amethod for treatment of an orthomyxovirus or paramyxovirus infection,comprising the step of administration to a subject in need thereof of aneffective amount of a compound of formula (I) as defined in claim
 1. 17.A method according to claim 16 in which the orthomyxovirus orparamyxovirus infection is an influenza A or B infection, parainfluenza,mumps or Newcastle disease.
 18. A method according to claim 16 in whichthe administration is to the respiratory tract by inhalation,insufflation or intranasally or a combination thereof.
 19. A method forthe detection of an orthomyxovirus or paramyxovirus infection whichcomprises the step of contacting the compound of formula (I) as definedin claim 1 with a sample suspected of containing the virus.
 20. A methodaccording to claim 16, further comprising the step of administering aneffective amount of one or more anti-viral agents used to treatrespiratory infections.